Hi everybody,
In my project I need to validate some microRNA in Treg cells.As the percentage of Treg cells inhuman is ~2% I am planning to isolate Cd4+Cd25+ Treg cells based on MACS® Separation and expand them by anti CD3 and CD28.
my concern is that expansion may affect the expression ratio of miRNA.
Would you please advise me regarding this issue?
Hi Fataneh Tavasolian
I would suggest to compare a Treg expansion vs. a Treg maintenance protocol in vitro using anti-CD3/CD28 (+ (recombinant) rIL-2) vs. Tregs maintained just by low dose rIL-2. Long term maintenance of Tregs in vitro without anti-CD3/CD28 stimulation will be difficult, but they should survive for a couple of days in culture. For protocols check out work from Alex Rudensky and Shimon Sakaguchi, who worked extensively on these topics. For a general overview and introduction check out:
https://www.nature.com/articles/s41392-017-0002-5
Article Development and Maintenance of Regulatory T cells
All the best & good luck with your experiments,
Michael
Hey,
I worked before with Treg cells and miRNA and from my expertise you should be able to isolate Treg cells via MACs, even with low number of cells you should be able to extract RNA and quantify microRNA. Consider to take Teff cells when you do your MACS sorting (Treg as CD4+CD25++CD127low and Teff as CD4+CD25- cells same apply for CD8Treg cells). Activating the cells will impact microRNA expression as some genes are known to regulate microRNA could be affected by the activation. Link for the paper : Article A microRNA profile of human CD8
Note also that microRNA expression is relative when you compare two population of cells.